Protein phosphorylation is one of the most common and extensively studied posttranslation modifications (PTMs). Phosphorylation and dephosphorylation of proteins control the function of the target protein, and their misregulation has been linked to many diseases, including cancer. Accordingly, protein kinases and phosphatases have emerged as important drug targets, boosted by the prominent success of Gleevec®, a tyrosine kinase inhibitor used to treat leukemia and other malignancies.
While most studies on protein phosphorylation have focused on the O-phosphorylation of Ser, Thr and Tyr residues, very little is known about histidine phosphorylation, which occurs at the imidazole nitrogens. The role of the histidine phosphorylation, however, is well documented in bacterial two-component signaling pathways; yet proteins with phosphohistidine (pHis) residues are also found in eukaryotic cells. In fact, in Physarum polycephalum, pHis accounts for 6% of the total phosphonoamino acids in its basic nuclear proteins. The prevalence of pHis is strikingly high among these proteins, considering that phosphotyrosine (pTyr) is found in less than 1% of eukaryotic cellular phosphoproteins.
Histidine phosphorylation plays an important role in cell processes. For example, histidine phosphorylation plays an important role in the signaling processes of higher eukaryotes. Further, the modification has been implicated in several processes, including G-protein signaling, ion conduction, secondary metabolism and chromatin biology. It is known that a histidine Kinase, nucleoside diphosphate Kinase, phosphorylates histidine and protein histidine phosphatase (PHP) dephosphorylates histidine. It has been found that histone (H4), which is one of the most abundant proteins in eukaryotic cells, and which, along with the core histones (H2A, H2B, and H3), forms the proteinaceous spool around which DNA is packaged in chromatin, is phosphorylated on histidine, located on positions 18 and 75 of H4. It has also been found that H4 histidine Kinase activity is about 400 times higher in human hepatocellular carcinoma liver tissue as compared to normal liver tissue. Thus, H4 histidine Kinase activity is strongly correlated with liver growth. Further, it has been found that mutation of His-18 (to either Ala or Gln) profoundly perturbed chromosome silencing in yeast and that this had the biggest effect on any of H4 mutants in silencing the yeast.
Despite these discoveries, further understanding of histidine phosphorylation has been impeded because it is technically challenging to detect and isolate pHis proteins. To further complicate matters, histidine can be phosphorylated in vivo at either of the nitrogen atoms in the imidazole ring, giving rise to two isomers, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Both isomers have a phosphoramidate (P—N) bond, which thermodynamically is higher in energy (Δ G° of hydrolysis: about −12 to about −14 kcal/mole) than the phosphoester bond of phosphohydroxyamino acids, as in pSer, pThr or pTyr (Δ G° of hydrolysis: about −6.5 to about −9.5 kcal/mole). Under acidic conditions, pHis is rapidly hydrolyzed due to protonation of the imidazole ring. Although pHis is stable under basic conditions, the instability of pHis under acidic conditions severely hampers detection and isolation of the pHis using standard protocols. Indeed, the instability of pHis in acidic conditions has impeded the development of antibodies to molecules containing pHis. For example, no specific antibodies against pHis-containing proteins have been found since immunogens with pHis will be dephosphorylated in serum.
Moreover, to further complicate matters, even though chemical phosphorylation of protein and peptides can be selectively carried out on histidine residues, it is difficult to obtain the 1-pHis form regioselectivitely, since 1-pHis isomerizes to the more thermodynamically stable 3-pHis under phosphorylation reaction conditions.
The present inventors, however, have found tools to investigate these problems. They have made pHis analogs which have been incorporated into peptides and proteins and have used these in the development of haptens and immunogens. The present inventors have prepared semi-synthetic pHis containing proteins and have generated pHis antibodies to further investigate the physiological functions of pHis.